Detection apparatus for biological materials and methods of making and using the same

ABSTRACT

Apparatus comprising a surface site layer having a distal site end, wherein the distal site end includes a substantially inorganic surface having a chemical composition selected from a group consisting of metals, semiconductors, insulators, and mixtures thereof, the surface positioned within a polypeptide bonding region and having a selective bonding affinity for a polypeptide; a plurality of interlayers between which the surface site layer is interposed, wherein the distal site end is distanced from the interlayers, first and second supports, wherein the surface site layer and the interlayers are interposed between the first and second supports; and first and second conductors provided on the first and second supports and having respective first and second distal conductor ends positioned within the polypeptide bonding region; wherein the conductors are capable of applying an external voltage potential across the polypeptide bonding region.

FIELD OF THE INVENTION

This invention relates to the field of apparatus for the detection,identification, characterization and further analysis of biologicalmaterials.

BACKGROUND OF THE INVENTION

Tremendous progress has been made over several decades in the study ofbiological materials ranging from amino acids, to proteins, to theentire human genome. In spite of the great strides that have alreadybeen made, cost-effective and timely analysis of biological materialsfrequently is still not a reality. For the myocardial infarction victimwaiting in the hospital emergency room or undergoing a heart bypassoperation, the time needed for conventional blood analysis in order todetect the telltale enzyme signature of a heart attack may be too long.Myriad other circumstances can be observed in which analytical testresults on biological materials simply take too long to generate, aren'tavailable where needed, and cost too much. Furthermore, conventionaldiagnostic tests typically are encumbered by their own particularcollection of analytical inadequacies, leading to false positive andnegative results at levels that are both intractable and statisticallysignificant.

Accordingly, there is a continuing need for analytical apparatus thatcan be used to detect, identify, characterize and otherwise analyzebiological materials, including for example amino acids and proteins.

SUMMARY OF THE INVENTION

Apparatus are provided comprising substantially inorganic surfacescomprising metals, semiconductors and/or insulators, which selectivelybond amino acids, polypeptides, proteins, and/or other substancescomprising amino acids. The selective bonding enables the detection,identification, and/or further analysis of the target aminoacid—comprising materials.

In an implementation, an apparatus is provided, including: a firstsurface site layer having a first distal site end, wherein the firstdistal site end includes a first substantially inorganic surface havinga first chemical composition selected from a group consisting of metals,semiconductors, insulators, and mixtures thereof, said first surfacepositioned within a polypeptide bonding region and having a selectivebonding affinity for a polypeptide; a plurality of first interlayersbetween which said first surface site layer is interposed, wherein thefirst distal site end is distanced from said first interlayers, firstand second supports, wherein said first surface site layer and saidfirst interlayers are interposed between said first and second supports;and first and second conductors provided on said first and secondsupports and having respective first and second distal conductor endspositioned within said polypeptide bonding region; said conductors beingcapable of applying an external voltage potential across saidpolypeptide bonding region.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a top view of an embodiment of an amino acid detection andidentification apparatus;

FIG. 2 shows an array of exemplary control test data for polypeptides,generated using the amino acid detection and identification apparatus ofFIG. 1;

FIG. 3 shows another array of exemplary control test data forpolypeptides, generated using the amino acid detection andidentification apparatus of FIG. 1;

FIG. 4 shows a further array of exemplary control test data forpolypeptides, generated using the amino acid detection andidentification apparatus of FIG. 1;

FIG. 5 shows data plotting the relative density of polar-basic,polar-acidic, polar-neutral and non-polar neutral polypeptides onvarious exemplary types of inorganic substrates;

FIG. 6 shows data plotting the adhered density of bound polypeptidesover a pH range;

FIG. 7 shows data plotting the adhered density of bound polypeptidesover a polypeptide concentration range;

FIG. 8 shows a schematic side view of an embodiment of an amino aciddetection and identification apparatus designed to selectively bond aparticular polypeptide;

FIG. 9 shows schematic side views of a progressive series of fourfurther amino acid detection and identification apparatus;

FIG. 10 shows data plotting the adhered density of bound polypeptidesversus separation between mutually adjacent AlGaAs apparatus layers;

FIG. 11 shows a schematic perspective view of an embodiment of an aminoacid detection and identification apparatus;

FIG. 12 shows a side view of an embodiment of another amino aciddetection and identification apparatus;

FIG. 13 shows a perspective view of the amino acid detection andidentification apparatus in FIG. 12;

FIG. 14 shows a side view of an embodiment of an apparatus embodyingmodifications of the apparatus shown in FIG. 12;

FIG. 15 shows an application of the apparatus shown in FIG. 12 for thedetection and identification of a target polypeptide macromolecule inwhich an antibody for the macromolecule is employed;

FIG. 16 shows a perspective view of an embodiment of an additional aminoacid detection and identification apparatus;

FIG. 17 shows steps of a method for making the amino acid detection andidentification apparatus of FIG. 1;

FIG. 18 shows steps of a method for using the apparatus of FIG. 1 fordetection and identification of an unknown polypeptide in a fluid;

FIG. 19 shows steps of a method for making the amino acid detection andidentification apparatus shown in FIGS. 8 and 9;

FIG. 20 shows steps of a method for using the apparatus of FIGS. 8 and 9for detection and identification of an unknown polypeptide in a fluid;

FIG. 21 shows steps of a method for making the amino acid detection andidentification apparatus shown in FIGS. 12, 13 and 14;

FIG. 22 shows steps of a method for using the apparatus of FIGS. 12, 13and 14 for detection and identification of an unknown polypeptidemacromolecule in a fluid; and

FIG. 23 shows steps of a method for making the amino acid detection andidentification apparatus shown in FIG. 16.

DETAILED DESCRIPTION

Apparatus are provided for the detection, identification,characterization, and other analysis of biological materials. Thebiological materials to be analyzed can include, for example, aminoacids, polypeptides, and proteins. The detection apparatus comprisedefined surfaces constituted by substantially inorganic materialsincluding metals, semiconductors, and/or insulators, to which biologicalmaterials selectively adhere in differential manners depending on thenatures of the particular surfaces and biological materials. Followingadhesion of biological materials to the apparatus, such materials can beoptically and electronically and otherwise analyzed in order to detect,identify and characterize the materials. By “substantially inorganic”herein is meant that the predominant components of the surfacecompositions do not comprise organic materials. However, it is to beunderstood that the incorporation of minor concentrations of organicmaterials that do not materially detract from the selective bondingaffinity of the substantially inorganic materials employed, is withinthe scope of these teachings. By “organic” is meant a compositioncomprising a carbon chain.

FIG. 1 shows a top view of an embodiment of an amino acid detection andidentification apparatus 100. The apparatus 100 is constituted by acolumn of test cells 102, 104, 106, 108, 110, 112, 114, 116 and 118. Inone embodiment, the test cells 102-118 are made from polished undopedGaAs wafers having a [100] orientation. The test cells 102-118 arecollectively capable of containing a sample of an amino acid orpolypeptide solution within a raised outer boundary wall 120, and ifdesired can be mutually separated by boundary walls 122, 124, 126, 128,130, 132, 134 and 136. The cells 102-118 have bottom surfaces 138, 140,142, 144, 146, 148, 150, 152 and 154 respectively, each comprising aselected inorganic metal, semiconductor, and/or insulator surface thatselectively adheres amino acids and polypeptides. In this embodiment,the metals palladium (Pd), gold (Au), titanium (Ti), platinum (Pt), andaluminum (Al); the semiconductors gallium-arsenide (GaAs), andaluminum-gallium-arsenide (AlGaAs); and the insulators silicon nitride(Si₃N₄), and silicon dioxide (SiO₂), were used. Accordingly, the bottomsurfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154 respectivelycomprise: GaAs, Si₃N₄, SiO₂, AlGaAs, Al, Pt, Ti, Au, and Pd. In oneembodiment, the AlGaAs was Al_(x)Ga_((1-x))As with x=about 0.3.

In one embodiment, optical characteristics of each of the bottomsurfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154 are recorded ascontrol data in the absence of a test solution. For example, the opticalcharacteristics can be determined using equipment suitable for detectingand recording the optical absorption and reflectance of each of thebottom surfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154. In thisregard, the apparatus 100 desirably includes test cells 102, 104, 106,108, 110, 112, 114, 116 and 118 arranged in a regular array. The testcells are carefully aligned for reading by corresponding equipmentsuitable for detecting and recording the optical absorption andreflectance of each of the bottom surfaces 138, 140, 142, 144, 146, 148,150, 152 and 154. It is understood that the vertical alignment of thetest cells 102, 104, 106, 108, 110, 112, 114, 116 and 118 in a column ismerely exemplary. For example, analogous test cell arrays can comprisehorizontal rows as well as multiple rows and columns, or other regulararrays such as test cells arranged in concentric circles. Test cells canalso be individually configured and analyzed.

A test solution comprising an unknown amino acid or polypeptide isapplied to the respective bottom surfaces 138, 140, 142, 144, 146, 148,150, 152 and 154 of the test cells 102, 104, 106, 108, 110, 112, 114,116 and 118. After allowing the passage of a suitable time period forany bonding of the test solution components on the bottom surfaces tooccur, such as about three (3) hours, the test solution is removed fromthe test cells 102-118 and the test cells are rinsed several times usinga test solution solvent. If present in the test solution, an unknownamino acid or polypeptide will selectively bond to some or all of thebottom surfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154. Theoptical characteristics of the apparatus 100 are then determined, usingthe same equipment for detecting and recording the optical absorptionand reflectance of each of the bottom surfaces. Changes in suchabsorption and reflectance on some or all of the bottom surfaces 138,140, 142, 144, 146, 148, 150, 152 and 154 are then computed bycomparison with the corresponding control data. Such changes in opticalabsorption and reflectance on the bottom surfaces 138, 140, 142, 144,146, 148, 150, 152 and 154 collectively constitute a signature foridentification of a particular amino acid or polypeptide present in thetest solution. For example, known samples of monomers or polypeptides ofeach of the twenty amino acids lysine (Lys), arginine (Arg), histidine(His), aspartic acid (Asp), glutamic acid (Glu), threonine (Thr), serine(Ser), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), proline (Pro),methionine (Met), cysteine (Cys), tryptophan (Trp), glycine (Gly),alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), andphenylalanine (Phe) can separately be subjected to these same steps.Such known polypeptides are commercially available, for example, fromAnaspec Inc., San Jose, Calif. These polypeptides can be made by solidstate synthesis. Background information is provided in Merryfield, R.B., J. Am. Chem. Soc., Vol. 85, pp. 2149+ (1963), the entirety of whichhereby is incorporated herein by reference. The resulting data can berecorded as unique signatures for each such amino acid or polypeptide. Atest solution comprising a given unknown amino acid or polypeptide canthen be identified by comparing the control signature data to test datacomputed on the unknowns using the apparatus 100. In another embodiment,all of the amino acid or polypeptide solutions can be tagged, such as byfluorescence, radioactivity, or ligands having known bonding activity.In the latter case, for example, bonding pairs such as biotin-avidin orantigen-antibody can be employed. The cells are then developed, such asby the measurement of fluorescence, radioactivity, or bonding affinitywith marked bonding pair counterparts, and the relative and absolutestrength of bonding in each test cell is read.

In one embodiment, the following test solution application procedure wasused. An apparatus 100 having a bottom surface with dimensions of about2 millimeters by 2 millimeters patterned on a GaAs substrate was placedin the respective test solution and left for about 3 hours. Theapparatus 100 was then removed from the test solution and rinsed indeionized water for 10 seconds and then dried in nitrogen gas.

There are four different classes of amino acids as determined by theirside chains, including polar-acidic, polar-basic, polar-neutral, andnon-polar neutral amino acids. The polar-acidic amino acids include Aspand Glu. The polar-basic amino acids include Lys, Arg and His. Thepolar-neutral amino acids include Thr, Ser, Asn, Gln, Tyr and Pro. Thenon-polar neutral amino acids include Met, Cys, Trp, Gly, Ala, Val, Ile,Leu and Phe. Desirably, each of these four groups of amino acids isconsidered to have bonding behavior on the bottom surfaces 138, 140,142, 144, 146, 148, 150, 152 and 154 that is somewhat consistent withinthe group. This consistency can aid in identification of test solutionscontaining unknown amino acids and polypeptides. In general, the polaramino acids, including the polar-acidic, polar-basic, and polar-neutralamino acids, are hydrophilic and accordingly may be soluble in polarsolvents which are used in the test solutions. For example, water can beused as the solvent. In general, the non-polar neutral amino acids arehydrophobic and accordingly may be soluble in nonpolar solvents such asnonpolar hydrocarbons. Polar amino acids may also be somewhat soluble innonpolar solvents, and nonpolar amino acids may also be somewhat solublein polar solvents.

The amino acid detection and identification apparatus 100 isparticularly suitable for the testing and identification of individualamino acids and polypeptides of individual amino acids. In general,solutions containing more than one amino acid are desirably separatedusing conventional techniques before amino acid identification using theamino acid detection and identification apparatus 100. Separation can becarried out, for example, using a chromatography column orelectrophoresis gel.

FIG. 2 shows an array 200 of exemplary control test data forpolypeptides of each of the 20 amino acids, generated using the aminoacid detection and identification apparatus 100. The polypeptides wereseparately prepared for each amino acid, generally having chain lengthsof ten (10) amino acid moieties except for minor concentrations ofpeptides having chain lengths of eight (8) amino acid moieties. Otherspecies may be present at insubstantial concentrations. Each polypeptidefurther included a 5-carboxyfluorescein (5F-AM) moiety bound at theexposed —N—H₂ group at the polypeptide end, leaving an exposed —C—O—O—H(carboxylic acid) group at the other end. Although the exposed —C—O—O—Hgroups are themselves reactive, this reactivity is overshadowed by thecomparatively greater cumulative reactivity of the various side chainsalso present in each amino acid moiety, of which there accordingly aregenerally eight (8) or ten (10) in each polypeptide. Therefore, bondingof the polypeptides to the substantially inorganic bottom surfacesoccurs through these side chains, a longitudinal side of the polypeptidethus being secured to the bottom surface. Although some end-bonding ofpolypeptides through the exposed —C—O—O—H groups may transiently occur,such bonding is disfavored due to entropy and other factors, andunlikely to persist. Since each polypeptide comprises such an exposed—C—O—O—H group, strong bonding there would lead to indistinguishableresults among testing of various polypeptides. Hence, operation of theamino acid detection and identification apparatus 100 takes advantage ofthe dynamics of this bonding environment to provide test resultsfacilitating differentiation between polypeptides of different aminoacids. In alternative embodiments, fluorescein, or fluorescein5-isothiocyanate (FITC), are used as markers instead of 5F-AM. In thecase of Cys polypeptides, the following peptide sequence was used inview of the potential for excessive disulfide crosslinking:5F-AM-Ala-Cys-Ala-Ala-Ala-Cys-Ala-Ala-Ala-OH. A potential source ofvariability in the results is the presence of contaminants in thepolypeptides that could induce or block adhesion to the substantiallyinorganic surfaces.

In this exemplary embodiment, each of the 20 polypeptides was separatelydissolved in water to generate the control solutions for testing. In oneembodiment, a 1.0 millimolar concentration of the polypeptides was used.The left-most column of FIG. 2 shows row headings for the control testdata array. The row headings identify and correspond to the metals,semiconductors and insulators on the bottom surfaces 138, 140, 142, 144,146, 148, 150, 152 and 154 respectively of the amino acid detection andidentification apparatus 100. The top-most row of FIG. 2 shows columnheadings for the control test data array. The column headings identifyand correspond to the individual known polypeptide solutions that wereseparately tested as reported in each column of the control test dataarray.

The control test data array shown in FIG. 2 is indicative of therelative and numerical concentrations of polypeptides bound to theindicated substantially inorganic surfaces when the test cells of theamino acid detection and identification apparatus 100 were subjected toknown aqueous solutions of each individual polypeptide. The units of thenumerical data are in 1×10³ polypeptides per square micrometer (μm²).The margin of error in the data was about twenty percent (20%). Thismargin of error included both statistical error and systematic error.Systematic errors include, for example, variations in results due todifferences in the processes for preparation of and of theconcentrations in the polypeptide solutions. The impact of margin oferror effects on the reliability and repeatability of test results canbe moderated by carrying out multiple trials and then averaging thenumerical results.

The control test cell data for the polar-acidic and polar-basicpolypeptides of Lys, Arg, His, Asp and Glu are grouped together in theleft section of the test cell data array shown in FIG. 2. The test celldata for the polar-neutral polypeptides of Thr, Ser, Asn, Gln, Tyr andPro are grouped together in the middle section of the test cell dataarray. The test cell data for the remaining non-polar neutralpolypeptides of Met, Cys, Trp, Gly, Ala, Val, Ile, Leu and Phe aregrouped together in the right section of the test cell data array. Eachof the data in the test cell data array visually and numericallyindicates the degree to which the designated polypeptide in each testbonded to the designated substantially inorganic surface. For example,data point 202 shows that an aqueous solution of polar-basic Argpolypeptide strongly bonded to Al, as indicated both by the dark shadingand the high numerical reading, 61×10³/μm². Data point 203 shows, incontrast, that the Lys polypeptide only lightly bonded to Al, asindicated both by the light shading and the light reading, 3.5×10³/μm².Further for example, data point 204 shows that an aqueous solution ofpolar-acidic Asp polypeptide firmly bonded to AlGaAs, as indicated bothby the medium dark shading and the elevated numerical reading,16×10³/μm². Data point 205 shows, in contrast, that the His polypeptideonly lightly bonded to AlGaAs, as indicated both by the light shadingand the light reading, 1.8×10³/μm². Additionally for example, data point206 shows that an aqueous solution of polar-neutral Thr polypeptidemoderately bonded to SiO₂, as indicated both by the grey shading and themoderate numerical reading, 5.2×10³/μm². Data point 207 shows, incontrast, that Asn polypeptide only minimally bonded to SiO₂, asindicated both by the lack of shading and the low numerical reading,0.9×10³/μm². Furthermore for example, data point 208 shows that anaqueous solution of non-polar neutral Gly polypeptide lightly bonded toSi₃N₄, as indicated both by the light shading and the light reading,3.4×10³/μm². Data point 209 shows, in contrast, that Ala polypeptideonly minimally bonded to Si₃N₄, as indicated both by the lack of shadingand the low numerical reading, less than (<) 0.5×10³/μm². In addition,for example, data point 210 shows that an aqueous solution of non-polarneutral Met polypeptide minimally bonded to Pt, as indicated both by thelack of shading and the low numerical reading, 0.7×10³/μm².

The visual and numerical test data reflected in the control test dataarray 200 can be used to identify the amino acid content of unknownaqueous polypeptide solutions. The control test data array in FIG. 2shows the strength of the bonding that results from exposure of each ofthe nine substantially inorganic surfaces separately to each of thetwenty amino acid oligomers (polypeptides). The strength of suchbonding, ranging from strong, to firm, moderate, light, and minimal,constitutes an indication of the amino acid identity as correlated withthe data in FIG. 2. FIG. 2 shows that most of the strongest bondingreactions occurred with polar-acidic and polar-basic polypeptides, andthat the strongest bonding reactions involved the Si₃N₄, SiO₂, AlGaAs,and Al surfaces. However, each of the control tests reported in thearray did generate a numerical bonding reading. In addition, each of theexemplary control tests reported in columns 212, 214 and 216 generated adifferent series of readings for the nine substantially inorganic testsurfaces. For example, Gly polypeptide in column 214 lightly bonded toSi₃N₄, SiO₂ and AlGaAs, but Ile polypeptide in column 216 lightly bondedonly to SiO₂ and AlGaAs, and instead minimally bonded to Si₃N₄. Thesedifferent series of numerical polypeptide bonding values can be used assignatures to distinguish Gly from Ile. Further analogous series ofnumerical bonding values can be used to identify other polypeptides in atest solution that is applied to the apparatus 100. The numericalbonding values for a given polypeptide are generally independent of theconcentration of the polypeptides in solution, provided that bondingsurface saturation by polypeptides occurs. Where, as reported in FIG. 2,oligomers of individual amino acids are tested, the test data reflectthe concentration of the polypeptides rather than of the individualamino acid molecules. The units are calibrated to 1×10³ amino acidoligomers per square micrometer.

FIG. 3 shows another array 300 of exemplary control test data foroligomers of the 20 amino acids prepared in the same manner as describedabove in connection with FIG. 2, generated using the amino aciddetection and identification apparatus 100. In this exemplaryembodiment, each of the twenty amino acid oligomers so tested wasconstituted in a 0.25 molar polypeptide solution using 1 Molar(N-2-[hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) (HEPES)diluted in water, in order to generate the control solutions fortesting. As in FIG. 2, the left-most column of FIG. 3 shows row headingsfor the control test data array. The row headings identify andcorrespond to the metals, semiconductors and insulators on the bottomsurfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154 respectively ofthe amino acid detection and identification apparatus 100. The top-mostrow of FIG. 3 shows column headings for the control test data array. Thecolumn headings identify and correspond to the individual knownpolypeptide solutions that were separately tested as reported in eachcolumn of the control test data array. The numerical data are againreported in units of 1×10³ amino acid oligomer molecules per squaremicrometer. The control test data array shown in FIG. 3 is indicative ofthe relative concentrations of polypeptides bound to the indicatedsubstantially inorganic surfaces when the test cells of the amino aciddetection and identification apparatus 100 were subjected to known HEPESsolutions of each individual polypeptide.

Each of the data in the test cell data array 300 visually andnumerically indicates the degree to which the designated polypeptide ineach test bonded to the designated substantially inorganic surface. Forexample, data point 302 shows that a HEPES solution of polar-basic Lyspolypeptide strongly bonded to Si₃N₄, as indicated both by the darkshading and the high numerical reading, 21×10³/μm². Data point 303shows, in contrast, that Ser polypeptide only lightly bonded to Si₃N₄,as indicated both by the light shading and the light reading,2.1×10³/μm². Further for example, data point 304 shows that a HEPESsolution of polar-neutral Thr polypeptide firmly bonded to SiO₂, asindicated both by the medium dark shading and the elevated numericalreading, 12×10³/μm². Data point 305 shows, in contrast, that Asnpolypeptide only lightly bonded to SiO₂, as indicated both by the lightshading and the light reading, 2.3×10³/μm². Additionally for example,data point 306 shows that a HEPES solution of non-polar neutral Metpolypeptide moderately bonded to Si₃N₄, as indicated both by the greyshading and the moderate numerical reading, 4.4×10³/μm². Data point 307shows, in contrast, that Gin polypeptide only lightly bonded to Si₃N₄,as indicated both by the light shading and the light reading,1.8×10³/μm². Furthermore for example, data point 308 shows that a HEPESsolution of non-polar neutral Gly polypeptide lightly bonded to SiO₂, asindicated both by the light shading and the light reading, 3.0×10³/μm².Data point 309 shows, in contrast, that Ala polypeptide only minimallybonded to SiO₂, as indicated both by the lack of shading and the lownumerical reading, <0.5×10³/μm². In addition, for example, data point310 shows that a HEPES solution of non-polar neutral Ala polypeptideminimally bonded to Al, as indicated both by the lack of shading and thelow numerical reading, 0.7×10³/μm². FIG. 3 shows the strength of thebonding that resulted from exposure of each of the nine substantiallyinorganic surfaces separately to each of the twenty amino acidoligomers. As in the case of FIG. 2, FIG. 3 shows that most of thestrongest bonding reactions occurred with polar-acidic and polar-basicpolypeptides, and that the strongest bonding reactions involved theSi₃N₄, SiO₂, AlGaAs, and Al surfaces. However, each of the test cellsdid generate a numerical data reading. In addition, each of theexemplary test data columns 312, 314, and 316 generated a differentseries of readings for the nine test surfaces. For example, Ilepolypeptide in column 314 moderately bonded to Al and lightly bonded toSiO₂, but Leu polypeptide in column 316 only lightly bonded to Al, andminimally bonded to SiO₂. These differential bonding patterns can beused to distinguish Ile from Leu. Further differential bonding patternspotentially can be mapped from the FIG. 3 data and used to distinguishany two HEPES amino acid oligomer solutions from each other in alikewise manner.

FIG. 4 shows a further array 400 of exemplary control test data foroligomers of the 20 amino acids prepared in the same manner as describedabove in connection with FIG. 2, generated using the amino aciddetection and identification apparatus 100. In this exemplaryembodiment, each of the twenty amino acid oligomers so tested wasconstituted in a 0.25 molar polypeptide solution using undiluteddimethyl sulfoxide (DMSO) in order to generate the control solutions fortesting. As in FIGS. 2 and 3, the left-most column of FIG. 4 shows rowheadings for the control test data array. The row headings identify andcorrespond to the metals, semiconductors and insulators on the bottomsurfaces 138, 140, 142, 144, 146, 148, 150, 152 and 154 respectively ofthe amino acid detection and identification apparatus 100. The top-mostrow of FIG. 4 shows column headings for the control test data array. Thecolumn headings identify and correspond to the individual known aminoacid oligomer solutions that were separately tested as reported in eachcolumn of the control test data array. The numerical data were againreported in units of 1×10³ amino acid oligomer molecules per squaremicrometer. The control test data array shown in FIG. 4 is indicative ofthe relative concentrations of polypeptides bound to the indicatedsubstantially inorganic surfaces when the test cells of the amino aciddetection and identification apparatus 100 were subjected to known DMSOsolutions of each individual polypeptide. In another embodiment,polypeptides were solubilized in a 1:5 DMSO:water solution at a 1millimolar polypeptide concentration. Higher concentrations of DMSO canbe beneficial in solubilizing polypeptides of Tyr, Phe and Leu.

Each of the data in the test cell data array 400 visually andnumerically indicates the degree to which the designated polypeptide ineach test bonded to the designated substantially inorganic surface. Forexample, data point 402 shows that a DMSO solution of polar-basic Argpolypeptide strongly bonded to Si₃N₄, as indicated both by the darkshading and the high numerical reading, 21×10³/μm². Data point 403shows, in contrast, that Thr polypeptide only lightly bonded to Si₃N₄,as indicated both by the light shading and the light reading,2.1×10³/μm². Further for example, data point 404 shows that a DMSOsolution of polar-acidic Glu polypeptide firmly bonded to AlGaAs, asindicated both by the medium dark shading and the elevated numericalreading, 10×10³/μm². Data point 405 shows, in contrast, that Asppolypeptide only lightly bonded to AlGaAs, as indicated both by thelight shading and the light reading, 3.7×10³/μm². Additionally forexample, data point 406 shows that a DMSO solution of polar-neutral Thrpolypeptide moderately bonded to Al, as indicated both by the greyshading and the moderate numerical reading, 4.4×10³/μm². Data point 407shows, in contrast, that Asn polypeptide only minimally bonded to Al, asindicated both by the lack of shading and the low numerical reading,0.7×10³/μm². Furthermore for example, data point 408 shows that a DMSOsolution of non-polar neutral Cys polypeptide lightly bonded to SiO₂, asindicated both by the light shading and the light reading, 3.4×10³/μm².Data point 409 shows, in contrast, that Met polypeptide moderatelybonded to SiO₂, as indicated both by the moderate shading and themoderate reading, 4.6×10³/μm². In addition, for example, data point 410shows that a DMSO solution of non-polar neutral Met polypeptideminimally bonded to Au, as indicated both by the lack of shading and thelow numerical reading, 0.9×10³/μm².

FIG. 4 shows the strength of the bonding that results from exposure ofeach of the nine substantially inorganic surfaces separately to each ofthe twenty amino acid oligomers. As in the case of FIGS. 2 and 3, FIG. 4shows that most of the strongest bonding reactions occurred withpolar-acidic and polar-basic amino acid oligomers, and that thestrongest bonding reactions involved the Si₃N₄, SiO₂, AlGaAs, and Alsurfaces. However, each of the test cells did generate a numericalreading. In addition, each of the exemplary test data columns 412, 414,and 416 generated a different series of readings for the nine testsurfaces. For example, Ala polypeptide in column 414 lightly bonded toSiO₂ and only minimally bonded to Si₃N₄, but Phe polypeptide in column416 lightly bonded to both SiO₂ and Si₃N₄. These differential bondingpatterns can be used to distinguish Ala from Phe. Further differentialbonding patterns can be mapped from the FIG. 4 data and used todistinguish any two aqueous amino acid oligomers from each other in alikewise manner.

The preceding discussion in connection with FIGS. 1-4 has been directedto substantially inorganic surfaces made from the metals Pd, Au, Ti, Pt,and Al; the semiconductors GaAs, and AlGaAs; and the insulators Si₃N₄,SiO₂. It is to be understood, however, that other metals, semiconductorsand insulators can be used in addition to or in substitution for one ormore of the substantially inorganic surfaces addressed in FIGS. 1-4. Inaddition, alloys or mixtures of two or more such metals, semiconductorsand insulators, and mixtures of one or more metals, semiconductors,and/or insulators can be used. Each of such materials will have its owncharacteristic pattern of bonding affinity for individual amino acidsand polypeptides. These bonding affinities can be mapped in the samemanner as discussed above in connection with FIGS. 1-4, and amino aciddetection and identification apparatus can be constructed and used inthe same manner.

In general, any metallic element or elements in the Periodic Table canbe used, alone or together with other metals, semiconductors, and/orinsulators, in a surface for selective amino acid or polypeptidebonding. In one embodiment, further metals that can be so used inaddition to Pd, Au, Ti, Pt, and Al include: magnesium (Mg), calcium(Ca), zirconium (Zr), vanadium (V), tantalum (Ta), chromium (Cr),molybdenum (Mo), tungsten (W), manganese (Mn), iron (Fe), cobalt (Co),nickel (Ni), copper (Cu), silver (Ag), zinc (Zn), cadmium (Cd), gallium(Ga), indium (In), thalium (Ti), tin (Sn), and lead (Pb).

In general, any substantially inorganic compound semiconductors can beused, alone or together with other substantially inorganic compoundsemiconductors, metals, and/or insulators, in a surface for selectiveamino acid or polypeptide bonding. The semiconductors can be doped asdesired, for example with elements that change the charge carriermobility of such semiconductors. In one embodiment, furthersubstantially inorganic compound semiconductors that can be so used, inaddition to GaAs and AlGaAs, include: indium phosphide (InP), indiumgallium arsenide (InGaAs), indium gallium phosphide (InGaAs), indiumgallium arsenide phosphide (InGaAsP), indium aluminum gallium arsenide(InAlGaAs), gallium nitride (GaN), indium nitride(InN), aluminum nitride(AlN), aluminum gallium nitride (AlGaN), indium aluminum gallium nitride(InAlGaN), gallium antimonide (GaSb), indium antimonide (InSb), aluminumantimonide (AlSb), aluminum gallium antimonide (AlGaSb), indium aluminumgallium antimonide (InAlGaSb), indium arsenic antimonide (InAsSb),gallium aluminum antimonide (GaAlSb), indium gallium antimonide(InGaSb), and gallium arsenic antimonide (GaAsSb).

In general, any substantially inorganic insulator can be used, alone ortogether with other insulators, metals, and/or semiconductors, in asurface for selective amino acid or polypeptide bonding. In oneembodiment, further substantially inorganic insulators that can be soused, in addition to Si₃N₄, and SiO₂, include: aluminum oxide (Al₂O₃),zinc oxide (ZnO), beryllium oxide (BeO), ferrite (Fe₃O₄), zirconiumoxide (ZrO₂), boron carbide (B₄C), silicon carbide (SiC), magnesiumdiboride (MgB₂), and in general, metallic oxides, carbides, borides,nitrides, and sulfides.

FIGS. 2, 3 and 4 respectively employed water, HEPES diluted in water,and DMSO as a solvent for the polypeptides, forming solutions of suchamino acid oligomers. Although the term “solution” is used throughoutthis discussion, it is to be understood that amino acid oligomers canalternatively be mobilized in other forms in fluids, such as, forexample, dispersions, suspensions, gels, emulsions, and aerosols.Furthermore, water, HEPES diluted in water, and DMSO are exemplarysolvents and fluid vehicles, and other solvents and fluid vehicles assuitable for the fluid mobilization of the polypeptides, proteins, orother amino acid—comprising compositions can also be used. Polarsolvents such as water preferably dissolve polar-acidic, polar-basic andpolar-neutral amino acids and polypeptides. Non-polar organic solventspreferentially dissolve non-polar neutral amino acids and polypeptides.

FIG. 5 shows summary bar graph data based on the tests carried out togenerate FIGS. 2-4, plotting the density on GaAs, Si₃N₄, SiO₂, Al and Pdsurfaces, of equivalent bound polypeptides×10³ per square micrometer(μm²), as to each of the twenty amino acid oligomers. FIG. 5 shows thatthe strongest bonding interactions occurred when Si₃N₄ and SiO₂ surfaceswere exposed to solutions of polar-basic polypeptides. FIG. 5 furthershows that polar-acidic polypeptides generally adhered strongly to Si₃N₄and SiO₂, although not as strongly as did the polar-basic polypeptides.FIG. 5 also shows that polar-basic and polar-acidic polypeptidesgenerally adhered firmly to Al, although not as strongly as to Si₃N₄ andSiO₂. FIG. 5 additionally shows that polar-neutral and non-polar neutralpolypeptides generally adhered moderately to Si₃N₄, SiO₂, and Al,although not as strongly as did polar-basic and polar-acidicpolypeptides. FIG. 5 furthermore shows that all types of polypeptides,including polar-basic, polar-acidic, polar-neutral, and non-polarneutral polypeptides, generally adhered at least minimally or lightly toGaAs and Pd. FIG. 5 makes clear that the relative bonding affinity ofpolypeptides to the five exemplary substantially inorganic surfaces canbe used in either a quantitative or relative qualitative manner togetherwith known controls in order to identify particular polypeptides insolution.

FIG. 6 shows graphed data plotting on the y-axis the adhered density, onSi₃N₄ surfaces, of equivalent bound polypeptides×10³ per μm² includingGlu, His, and Lys, and on the x-axis a pH range of between about 5.5 andabout 11.75. The pH can be increased, for example, by addition of NH₄OH.As to Glu, the density of equivalent bound polypeptides remained stableat about 27,000 per μm² of surface across a pH range of between about5.5 and about 6.0; and gradually dropped to a minimal density that wasthen maintained at a pH above about 6.2. As to His, the density ofequivalent bound polypeptides remained stable at about 22,000 per μm² ofsurface across a pH range of between about 5.2 and about 7.2; andgradually dropped to a minimal density that was then maintained at a pHabove about 7.3. As to Lys, the density of equivalent bound polypeptidesremained stable at about 28,000 per μm² of surface across a pH range ofbetween about 6.2 and about 10.0; and gradually dropped to a minimaldensity then maintained at a pH above about 10.6. Similar effects can bedemonstrated for the other polar-charged polypeptides, includingpolar-basic Arg and polar-acidic Asp. Ionization behavior of thepolar-neutral polypeptides can also be used to affect the bondingaffinity of these amino acid oligomers to substantially inorganicsurfaces.

FIG. 7 shows graphed data plotting on the y-axis the adhered density onSi₃N₄ surfaces, of equivalent bound His polypeptides×10³ per μm², and onthe x-axis a polypeptide concentration range of between about 1×10⁻⁴millimolar (thousandths of a mole of polypeptides per liter) (mM/l) andabout 1×10⁻¹ mM/l. All concentrations are expressed as equivalentpolypeptides in millimoles. FIG. 7 shows that for polypeptide solutionshaving amino acid oligomer concentrations of less than about 5×10⁻³mM/l, no appreciable bonding to the substantially inorganic surfacesoccurs. Over a concentration range of between about 5×10⁻³ mM/l andabout 1×10⁻² mM/l, the density of adhered polypeptides steadilyincreases to about 24,000 equivalent bound polypeptides per μm², andthis density is then maintained at higher polypeptide concentrations.Hence, FIG. 7 shows that a given substantially inorganic surface has alimited capacity for bonding polypeptides before saturation occurs.

FIG. 8 shows a schematic side view of an embodiment of an amino aciddetection and identification apparatus 800 designed to selectively bonda particular polypeptide, and shows such a polypeptide 802 suspendedover the apparatus 800. In this embodiment, the apparatus 800 isdesigned to selectively bond a polypeptide comprising five (5) Aspmolecules forming a polypeptide chain indicated at 806, the polypeptidehaving either three (3) or five (5) Leu molecules further extending thepolypeptide chain at both ends. The amino acid detection andidentification apparatus 800 comprises a midlayer of AlGaAs 805interposed between two layers of GaAs 810 and 815. The GaAs layers 810and 815 are visually distinguished by cross-hatching. The thickness ofthe AlGaAs midlayer 805 is about 1.9 nanometers (nm) as indicated by thedouble arrow 820. This thickness is less than or equal to thelongitudinal length of the polypeptide consisting of five Asp moleculesindicated at 806.

In one embodiment, the thickness of the GaAs layer 810 is about 1.7 nmas indicated by the double arrow 825. This thickness approximatelymatches or exceeds the longitudinal length of a polypeptide 826consisting of three Leu molecules having end-bonded 5-carboxyfluorescein(5F-AM). Fluorescein is a fluorescent marker that enables detection of abound polypeptide on the amino acid detection and identificationapparatus 800. The 5F-AM marker extends the longitudinal length of thepolypeptide itself. The thickness of the GaAs layer 815 is about 1.2 nmas indicated by the double arrow 830. This thickness approximatelymatches or slightly exceeds the longitudinal length of a polypeptide 827consisting of three Leu molecules.

In an alternative embodiment, the thickness of the GaAs layer 810 isabout 2.5 nm as indicated by the double arrow 825. This thicknessapproximately matches or exceeds the longitudinal length of apolypeptide consisting of five Leu molecules 826 having end-bonded5F-AM. The thickness of the GaAs layer 815 is about 2 nm as indicated bythe double arrow 830. This thickness approximately matches or exceedsthe longitudinal length of a polypeptide 827 consisting of five Leumolecules.

In a further alternative embodiment, the AlGaAs midlayer 805 is extendedto include region 807 indicated by a dotted line in FIG. 8. In thisembodiment, the double arrow 821 indicates the distance j between theAlGaAs midlayer 805 and the polypeptide 806 comprising five (5) Aspmolecules. Additionally in this embodiment, the double arrow 822indicates the distance k between the GaAs layer 810 and the polypeptideconsisting of five Leu molecules 826 having end-bonded 5F-AM. Further inthis embodiment, the double arrow 823 indicates the distance l betweenthe GaAs layer 815 and the polypeptide consisting of five Leu molecules827 having an exposed —C—O—O—H end group. It can be seen that thedistance j is less than the distances k and l. Stated otherwise, theregion 807 constitutes a shelf on which the polypeptide 806 can rest.This shelf, and the greater distances k and l, provide extra space onwhich the polypeptides 826 and 827 can rest when the polypeptide 806becomes bonded to region 807 of the AlGaAs midlayer 805. In this manner,the polypeptide 806 can more readily bond to the AlGaAs midlayer 805without steric hindrance between the side groups of the Leu moieties andthe GaAs layers 810 and 815.

In use, the amino acid detection and identification apparatus 800 isexposed to a solution of polypeptides. If any of the polypeptidesinclude chains comprising Leu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu,or a similar polypeptide with two additional Leu molecules at each endof the chain, then such polypeptides will selectively bond to the aminoacid detection and identification apparatus 800 having layers 805, 810and 815 of the appropriate thickness discussed above. This bonding willoccur in alignment with the arrows 816, 817, 818 and 819. Polypeptideshaving additional chain portions beyond the selectively bondedpolypeptide will adhere only such selected polypeptide, leaving theother chain portions unbonded and trailing away from the apparatus 800.Selective polypeptide bonding can be detected, for example, by labelingthe polypeptides with 5F-AM as discussed earlier. It will be understoodthat a given apparatus 800 may be capable of bonding more than onespecific polypeptide sequence, as the various bonding surface materialsoften have bonding affinities for more than one amino acid.

The term “layer” as used throughout this specification is defined as abody of the subject material as applied over an adjoining surface,however such body is formed. A “layer” may have a non-uniform thickness,does not have to be completely continuous, and may be the result of anydesired deposition process undertaken in one or more than one steps.Hence, a “layer” may also comprise multiple layers of the same ordifferent materials, which may or may not interpenetrate each other, andwhich layers together are referred to as the “layer”. There is noparticular limitation on the thickness of a layer except as stated.

FIG. 9 schematically illustrates the importance of controlling thethicknesses of the layers in the amino acid detection and identificationapparatus 800, in order to maximize the bonding potential for thepolypeptide 802. The four images 900 represents schematic side views ofa progressive series of amino acid detection and identificationapparatus 905, 910, 915 and 920 similar to the amino acid detection andidentification apparatus 800. Regions in the apparatus 905, 910, 915 and920 distinguished by cross-hatching indicate GaAs layers 902, which areinterposed by AlGaAs layers 904 without cross-hatching, similar to thestructure of the apparatus 800 shown in FIG. 8. Each of the apparatus905, 910, 915 and 920 has more than one bonding site forLeu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu, or for a similarpolypeptide with two additional Leu molecules at each end of the chain.In the apparatus 905, the GaAs layers between any two adjacentpolypeptide bonding sites have a thickness d, indicated by the doublearrow 925. The thickness d is more than adequate to prevent stericinterference between adjacently bonded polypeptides. In the apparatus910, the GaAs layers between any two adjacent polypeptide bonding siteshave a thickness e indicated by the double arrow 930 that is stilladequate to prevent steric interference between adjacently bondedpolypeptides, but some of the polypeptides nearly butt ends as atexemplary point 935. In the apparatus 915, the GaAs layers between anytwo adjacent polypeptide bonding sites have a thickness f indicated bythe double arrow 940 that is too small to prevent steric interferencebetween adjacent polypeptides at some of the potential bonding sites.Although some bonding can still occur, exemplary bonding sites 945 and950 are sterically blocked, reducing the selective bonding capacity ofthe apparatus 915. In the apparatus 920, the distance between the AlGaAslayers of any two adjacent polypeptide bonding sites constitutes athickness g indicated by the double arrow 955 that is too small to allowany selective bonding of polypeptides. At every intended bonding site,steric interference between adjacent AlGaAs midlayers prevents bondingof any polypeptides at any of the intended bonding sites. FIG. 9illustrates the importance of designing and controlling the thicknessesof substantially inorganic layers for selective bonding of polypeptidesin order to avoid decreased apparatus capacity or total failure due tosteric hindrance factors. In a further alternative embodiment, theAlGaAs midlayers are extended in the same manner as discussed above inconnection with the midlayer 805 and region 807 shown in FIG. 8.

FIG. 10 plots data regarding the bonding of polypeptides on the aminoacid detection and identification apparatus 905-920 shown in FIG. 9. Thex-axis denotes the separations d, e, f and g, in nm, between mutuallyadjacent AlGaAs layers as shown in FIG. 9. The y-axis denotes theadhered density of equivalent bound polypeptides×10³ per μm² on theamino acid detection and identification apparatus 905-920. The circulardata plot trials in which the polypeptides applied to the amino aciddetection and identification apparatus 905-920 had three Leu moieties onthe polypeptide ends, each having an end-to-end length of about 1.2 nm.The square data plot trials in which the polypeptides applied to theamino acid detection and identification apparatus had five Leu moietieson the polypeptide ends, each having an end-to-end length of about 2 nm.In each case, the polypeptides were labeled at one end with 5F-AM.Referring first to the circular data points, the adhered density ofLeu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu polypeptides steadilyincreased as the separation between the AlGaAs layers was reduced fromabout 3.4 nm to about 2.5 nm to about 1.4 nm, and then dramaticallydropped as the separation was reduced to about 1.2 nm. The steadyincrease evident in the first three such data points indicates thatthere was no significant steric hindrance to polypeptide binding, whilethe packing density of binding sites became correspondingly greater. Thesudden drop in polypeptide adhesion density at an AlGaAs layerseparation of about 1.2 nm indicates that substantial steric hindranceto polypeptide bonding arose at this smaller layer separation. A similarpattern resulted in the square data points regarding the adhered densityof Leu-Leu-Leu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu-Leu-Leu. In eachcase, the polypeptides were labeled at one end with 5F-AM. There, thepolypeptide adhesion density increased as the AlGaAs layer separationwas decreased from about 3.4 nm to about 2.5 nm. However, the adhesiondensity at an AlGaAs layer separation of about 1.4 nm was almost as lowas that at a separation of about 1.2 nm. This result is consistent withthe additional chain length of the polypeptides being selectively bondedin the square plotted data, because the additional polypeptide lengthcaused steric hindrance to first arise at a greater AlGaAs layerseparation. The results of the trials reported in FIG. 10 furtherillustrate the trend in bonding performance through the progression inamino acid detection and identification apparatus 905, 910, 915 and 920,as discussed in connection with FIG. 9.

FIG. 11 shows a schematic perspective view of an embodiment of an aminoacid detection and identification apparatus 1100. The apparatus 1100comprises three sandwiched substantially inorganic layers 1105, 1110 and1115, each of which may be independently selected from among thesubstantially inorganic metals, semiconductors and/or insulators andmixtures as earlier discussed. The optimal compositions of the layers1105, 1110 and 1115 are determined by the polypeptide to be selectivelybonded to and thus made detectable by the apparatus 1100. Referring backto FIG. 8, the selected polypeptide will then bond across layers 1105,1110 and 1115 in the same manner as the exemplaryLeu-Leu-Leu-Asp-Asp-Asp-Asp-Asp-Leu-Leu-Leu polypeptide bonded acrosslayers 805, 810 and 815 discussed in connection with FIG. 8. Oneadvantage of the structure of the apparatus 1100 is that the layers1105, 1110 and 1115 can be successively built up on a non-bondingsubstrate 1120, and then etched to reveal layers with the selectedbonding activity. In one embodiment, the thus exposed layers 1105, 1110and 1115 are mutually flush so that a selected polypeptide will bond toall three layers. In another embodiment, one or more of the layers maybe recessed or formed from a non-bonding material that serves as aspacing element rather than a bonding surface. In one embodiment, onlythe layer 1110 serves as a bonding surface for amino acids. In onemodification of that embodiment, conductors 1125 and 1130 are inelectrical communication with an external source for applying a voltagepotential across the layer 1110. In this manner, a change inconductivity across the layer 1110 detected by the external voltagesource is an indication of selective bonding of amino acids orpolypeptides on the layer 1110. Although the exemplary apparatus 1100comprises three layers 1105, 1110 and 1115, any desired number ofbonding and/or spacing layers can be built up and exposed to selectivelybond a desired polypeptide sequence. It will be understood that a givenapparatus 1100 may be capable of bonding more than one specificpolypeptide sequence, as the various surface materials often havebonding affinities for more than one amino acid. Apparatus can bedesigned to carry out the same operations with regard to macromoleculescomprising amino acids, such as proteins.

FIG. 12 shows an embodiment of an amino acid detection andidentification apparatus 1200 which is suitable for the detection andidentification of macromolecules comprising amino acids. The apparatus1200 comprises AlGaAs surface site layers 1205, 1210, and 1215interposed between GaAs interlayers 1220, 1225, 1230 and 1235. Distalsite ends 1240, 1245 and 1250 of the AlGaAs surface site layers 1205,1210, and 1215 extend beyond ends of the GaAs interlayers 1220, 1225,1230 and 1235, forming a polypeptide bonding region 1227. All of theforegoing layers are sandwiched between AlGaAs support layers 1255 and1260. Conductors 1265 and 1270 are provided on surfaces of the AlGaAssupport layers 1255 and 1260 adjacent to the polypeptide bonding region1227. The AlGaAs support layers 1255 and 1260 serve to position theconductors 1265 and 1270 adjacent to the polypeptide bonding region1227, and to form the bonding region 1227 as a well for containing atest solution potentially containing a polypeptide macromolecule. Atarget polypeptide macromolecule 1285 having regions that selectivelybond with AlGaAs, desirably located in precise alignment with the distalsite ends 1240, 1245 and 1250, will then selectively bond on surfaces ofthe distal site ends to the apparatus 1200. The conductors 1265 and 1270may be in electrical communication with an external voltage source forapplying a potential across the bonding region 1227 between distal ends1275 and 1280 of the conductors 1265 and 1270 respectively, forconfirming the presence of a selectively bound polypeptide macromoleculeon the apparatus 1200. A change in the conductivity across the bondingregion 1227 is an indication of such presence. Alternatively, forexample, the conductors 1265 and 1270 can be substituted by opticalwaveguides such as optical fibers or optical planar waveguides mutuallyaligned for light transmission and directing light across the bondingregion 1227 so that a change in transmitted light through such opticalwaveguides is an indication of the selective bonding of a polypeptidemacromolecule. In use, a solution potentially comprising the targetpolypeptide macromolecule is placed in the vicinity of the region 1227.If present in the solution, a target polypeptide macromolecule 1285 thenselectively bonds to the apparatus 1200. FIG. 13 shows the sameapparatus 1200 in perspective view. FIG. 13 shows the location of theconductors 1265 and 1270, and exemplary AlGaAs surface site layers 1205and 1210.

FIG. 14 shows an apparatus 1400 embodying modifications of the apparatus1200. The modifications enable the controlled and independentapplication of two different voltages to precisely located regions of aselectively bound polypeptide macromolecule 1285. In this embodiment,each of the AlGaAs surface site layers 1205, 1210 and 1215 is formedfrom an electrical conductor or semiconductor having a charge carriermobility, optionally p-doped or n-doped. The layers 1205 and 1215 are inelectrical contact with conductor 1290, and the layer 1210 is inelectrical contact with conductor 1295. Accordingly, a first voltage V1can be applied to the layers 1205 and 1215 through conductor 1290, and asecond voltage V2 can independently be applied to the layer 1210 throughconductor 1295. Application of such voltages can be used to modulate thebinding of the target polypeptide macromolecule as a further aid in itsdetection and/or identification.

FIG. 15 shows an application of the apparatus 1200 for the detection andidentification of a target polypeptide macromolecule 1285, employing anantibody to the target polypeptide macromolecule. In this embodiment, anantibody 1297 shown in region “B” is provided having specific bindingaffinity for a target polypeptide macromolecule 1285 constituting anantigen. The antibody 1297 is anchored within the region 1227 by apolypeptide chain 1298 shown in region “A”. The polypeptide chain 1298is selectively bonded to exemplary layers 1205 and 1210. The polypeptidechain 1298 is bonded to the antibody 1297 at their interface asindicated by the dotted line 1299. The polypeptide chain 1298 comprisesa polypeptide subregion having a specific binding affinity for theexemplary layers 1205 and 1210. In use, the antibody is selectivelybonded to the region 1227 by the layers 1205 and 1210. A solution thatpotentially includes the target polypeptide macromolecule 1285 is thenplaced in the vicinity of the region 1227. If the target polypeptidemacromolecule 1285 is present in the solution, a target macromolecule1285 selectively bonds to the antibody 1297 secured to the region 1227of the apparatus 1200.

FIG. 16 shows an embodiment of an additional amino acid detection andidentification apparatus 1600 which is suitable for the selectivedetection and identification of an amino acid, polypeptide, ormacromolecule comprising amino acids. The apparatus 1600 comprises anelectrically conductive comb 1602 comprising tines 1604, 1606, 1608,1610, 1612, 1614, 1616 and 1618. The apparatus 1600 further comprises anelectrically conductive comb 1603 comprising tines 1605, 1607, 1609,1611, 1613, 1615, 1617, 1619, and 1621. The tines 1604-1618 of the comb1602 are interlaced with and separated by small distances from the tines1605-1621 of the comb 1603. Pad 1630 is in electrical communication withthe comb 1602; and pad 1632 is in electrical communication with the comb1603. The pads provide a surface of adequate size for the application ofan externally generated voltage potential, such as by touchingelectrically charged probes to the pads. The combs 1602 and 1603 aremade from two independently selected electrically conductive materialscomprising substantially inorganic metals, semiconductors and/orinsulators as earlier discussed. Although the combs are not fabricatedsolely from insulators, they can be fabricated from materials comprisinginsulators together with metals and/or semiconductors. The combs 1602and 1603 are separated by substrate 1634. The electrical conductivity ofthe substrate 1634 is adequately reduced relative to that of the combs1602 and 1603 such that a voltage potential across a gap between thecombs 1602 and 1603 can be generated. The small distances between thetines of the combs are then designed and precisely fabricated in amanner analogous to the manner in which the AlGaAs layer 805 is preparedas discussed above in connection with FIGS. 8-10. In use, a solution ofan amino acid, polypeptide, or macromolecule comprising amino acids isapplied to the tines 1604-1621 of the combs 1602 and 1603 and to the gapbetween them across the substrate 1634. In one embodiment, the minimumpath length across the gap is within a range of between about 200Angstroms (Å) and about 2000 Å. Target amino acids, polypeptides, ormacromolecules with a bonding affinity for the alternating surfaces ofthe tines in combs 1602 and 1603 will selectively bond to the apparatus1600. Following removal of the solution of unbonded amino acids,polypeptides or other macromolecules, the charge carrier conductivity ofthe combs 1602 and 1603 can be tested. A change in conductivityindicates bonding of the amino acids, polypeptides, or macromolecules.

FIG. 17 shows an embodiment of a method 1700 for making the amino aciddetection and identification apparatus 100 as discussed above inconnection with FIGS. 1-4. In a series of steps 1705 and 1710, the aminoacid detection and identification apparatus 100 is fabricated. In step1705, a substrate is provided for a column of test cells 102, 104, 106,108, 110, 112, 114, 116 and 118. The substrate may be made from anymaterial suitable for the fabrication of a supportive base for testsurfaces, such as a polymer, metal, or ceramic. A raised outer boundarywall 120 is provided on the substrate that is capable of containing asample of an amino acid or polypeptide solution. Further raised boundarywalls 122, 124, 126, 128, 130, 132, 134 and 136 are provided on thesubstrate, defining and mutually separating the test cells 102-118. Thecells 102-118 define exposed and mutually separated portions of thesubstrate. In step 1710, bottom surfaces 138, 140, 142, 144, 146, 148,150, 152 and 154, respectively, are provided on each of the exposed andmutually separated portions of the substrate. The bottom surfaces138-154 each independently comprise a selected inorganic metal,semiconductor, and/or insulator surface that selectively adheres aminoacids. The bottom surfaces 138-154 can each be prepared on the substrateusing any suitable process, such as evaporation, vapor deposition orelectrodeposition. In this embodiment, the metals Pd, Au, Ti, Pt, andAl; the semiconductors GaAs and AlGaAs; and the insulators Si₃N₄ andSiO₂, are used. Accordingly, the bottom surfaces 138, 140, 142, 144,146, 148, 150, 152 and 154 respectively comprise: GaAs, Si₃N₄, SiO₂,AlGaAs, Al, Pt, Ti, Au, and Pd. In one embodiment, oxides naturallyformed on the metal surfaces such as aluminum oxide are not removed.

FIG. 18 further shows steps 1805, 1810, 1815, 1820, and 1825 of a method1800 for using the apparatus 100 for detection and identification of apolypeptide 1830 in a fluid. In step 1805, a selected controlpolypeptide composition is deposited in each of the test cells 102, 104,106, 108, 110, 112, 114, 116 and 118 of the amino acid detection andidentification apparatus 100. As earlier discussed, the polypeptidecompositions can be mobilized in any form of fluid, such as, forexample, solutions, dispersions, suspensions, gels, emulsions, andaerosols. Furthermore, solvents and fluid vehicles other than water,HEPES diluted in water, and DMSO can be used. In step 1810, first dataare recorded as to selective affinity of each selected test surface foreach control polypeptide composition. A separate apparatus 100 may beused to carry out each control test, or an apparatus 100 can bechemically treated to remove any bound polypeptides and reused. In step1815, an unknown polypeptide composition is deposited on an amino aciddetection and identification apparatus 100. In step 1820, second dataare recorded as to selective affinity of each selected test surface forthe unknown polypeptide composition. In step 1825, the second data arecorrelated with the first data to detect and identify the unknownpolypeptide composition 1830. It is to be understood that thecompositions can comprise more than one polypeptide or may have beenpreviously treated, e.g., by chromatography or electrophoresis, toisolate a single polypeptide for identification.

FIG. 19 shows an embodiment of a method 1900 for making amino aciddetection and identification apparatus 800 as discussed above inconnection with FIGS. 8 and 9. In a series of steps 1905, 1910, 1915,and 1920, the amino acid detection and identification apparatus 800 isfabricated. In step 1905, a first plurality of surface sites 810 areprovided, each comprising a first substantially inorganic surfaceselected from the group consisting of metals, semiconductors,insulators, and mixtures, each of said first surfaces having selectiveaffinity for bonding of a portion of a polypeptide. In step 1910, asecond plurality of surface sites 805 are provided, each comprising asecond substantially inorganic surface selected from the groupconsisting of metals, semiconductors, insulators, and mixtures, each ofsaid second surfaces having selective affinity for bonding of a portionof a polypeptide. In another embodiment, in step 1915, a third pluralityof surface sites 815 are provided, each comprising a third substantiallyinorganic surface, the second plurality of surface sites 805 interposedbetween and adjacent to the first and third pluralities 810 and 815, andhaving a thickness for spacing the first and third pluralities ofsurface sites apart by a distance suitable for selectively bondinganother portion of a polypeptide to the second plurality of surfacesites 805. The first, second and third pluralities of surface sites 805,810 and 815 can be fabricated, for example, as successively depositedlayers on a substrate, not shown, which can for example be interfacedwith the side 812 of the layer 810. In one embodiment, in step 1920 eachof the first plurality of surface sites 810 is provided with a firstsubstantially inorganic surface extending for a distance away from theadjacent second surface site 805, the distance being suitable forselectively bonding a portion of a polypeptide to each of the firstplurality of surface sites 810.

The deposition steps in FIG. 19 can be carried out, for example, using avapor deposition process such as molecular beam epitaxy (MBE). Othervapor deposition techniques, such as plasma enhanced chemical vapordeposition (PECVD) can also be used. The AlGaAs layers can beselectively exposed using an etch of H₂O₂/NH₄OH followed by cleaningwith an oxygen plasma. In one embodiment, the AlGaAs surface site layershad thicknesses of about 0.85 nm and the GaAs interlayers hadthicknesses within a range of between about 1.15 nm and about 5.94 nm.To achieve a total device thickness of about 10 microns, roughly 3,000alternating periods of GaAs and AlGaAs could be used. Extensive pausesbetween depositions of the alternating periods are advantageously used.After fabrication of the device periods, the layering is exposed bycleavage in air. A wet etch of H₂O₂/NH₄OH (500:1) is then applied,followed by a water rinse and nitrogen drying. The wet etch is selectiveto GaAs versus AlGaAs by a factor of at least about 300:1, thus leavinga set of veins of AlGaAs surface sites protruding above a background ofGaAs. Exposure to water modifies the adhesive properties of AlGaAs whencompared to washes only in organic solvents. In an alternativeembodiment, AlGaAs is selectively applied to a GaAs substrate usinglithographic masking techniques to form the veins. In anotherembodiment, the Al content of AlGaAs is used to control the etching. Asthe Al content is reduced, the etching activity on the AlGaAs itselfincreases, enabling reduction of the AlGaAs thickness.

In one embodiment, Si₃N₄ and SiO₂ were deposited as 30 nanometer (nm)thick films using plasma enhanced chemical vapor deposition (PECVD).Photolithography was used to produce patterns on a micron-length scale,and dry reactive ion etching (RIE) of the Si₃N₄ and SiO₂ wasaccomplished with CF₄ and CH₃F respectively to reveal the underlyingGaAs. The metals Au, Pd, Pt, Ti and Al were deposited using electronbeam- or thermal-evaporation. The apparatus 800 were exposed to a four(4) minute oxygen plasma etch as a cleaning step.

FIG. 20 further shows steps 2005, 2010 and 2015 of a method 2000 forusing the apparatus 800 for detection and identification of apolypeptide 2020 in a fluid. Referring to FIG. 20, the amino aciddetection and identification apparatus 800 is first calibrated withknown polypeptides in step 2005. Next, data are recorded in step 2010 asto selective affinity of the apparatus 800 for an unknown polypeptidecomposition. In step 2015, the experimental data are correlated with thecalibration data to detect and identify the polypeptide 2020.

FIG. 21 shows an embodiment of a method 2100 for making amino aciddetection and identification apparatus 1200 as discussed above inconnection with FIGS. 12, 13 and 14. In a series of steps 2105, 2110 and2115, the amino acid detection and identification apparatus 1200 isfabricated. In step 2105, a first surface site layer 1205 is provided,formed from a first substantially inorganic composition selected fromthe group consisting of metals, semiconductors, insulators, andmixtures, said first surface site layer having selective affinity forbonding of a polypeptide. In step 2110, a plurality of interlayers 1220and 1225 are provided, between which the first surface site layer 1205is interposed. In step 2115, the first surface site layer 1205 isprovided with a distal site end 1240 extending away from the interlayers1220 and 1225, the distal site end 1240 comprising a first surfacehaving selective affinity for bonding of a polypeptide. In oneembodiment, steps 2105, 2110 and 2115 are repeated for the fabricationof a second surface site layer 1210. In another embodiment, the firstand second surface site layers 1205 and 1210 and their respectiveinterlayers are interposed between first and second support layers 1255and 1260 in step 2120, and first and second conductors 1265 and 1270 areprovided on said first and second support layers in step 2125 includingfirst and second distal conductor ends 1275 and 1280 mutually aligned ata gap adjacent to said first and second surface sites. Referring back toFIG. 14, in an alternative embodiment each of the AlGaAs surface sitelayers 1205, 1210 and 1215 is formed from an electrical conductor orsemiconductor, optionally p-doped or n-doped. The layers 1205 and 1215are then suitably fabricated by semiconductor masking, deposition andetching steps so as to be placed in electrical contact withsubsequently-formed conductor 1290. Similarly, the layer 1210 issuitably fabricated by semiconductor masking, deposition and etchingsteps so as to be placed in electrical contact with conductor 1295. Forexample, in step 2130 metals suitable for forming conductors compatiblewith the n- and p-doped semiconductors used in making a particulardevice can be diffused through the various layers to make selectivecontact with the layers 1205, 1210 and 1215.

FIGS. 22 further shows steps 2205, 2210, 2215, 2220, 2225 and 2230 of amethod 2200 for using the apparatus 1200 for detection andidentification of a polypeptide macromolecule 1285 in a fluid. In step2205 a control polypeptide macromolecule is deposited at the firstsurface site 1240. In step 2210, first data are recorded as to selectiveaffinity of the first surface site 1240 for the control polypeptidemacromolecule. In step 2215, the apparatus 1200 is regenerated byremoval of any bound control polypeptide, or an additional amino aciddetection and identification apparatus is provided for testing of afluid comprising an unknown polypeptide macromolecule. In step 2220, anunknown polypeptide macromolecule is deposited at the first surface site1240. In step 2225, second data are recorded as to selective affinity ofthe first surface site 1240 for the unknown polypeptide macromolecule.In step 2230, second data are correlated with first data to detect andidentifY the polypeptide macromolecule 2235. In an embodiment where theconductors 1290 and 1295 are provided, an external bias can be providedin electrical communication with such conductors, capable of applying avoltage potential across the region 1227.

In one embodiment, the specificity of an apparatus 1200 for bonding andthus detecting a specific polypeptide macromolecule is tested by firstproducing polypeptides having active regions consistent with the activeregions of the macromolecule considered as bonding sites, which arefluorescently labeled. The capability of the apparatus 1200 to bond suchpolypeptides is then tested. When a suitable spatial arrangement ofbonding sites in the apparatus 1200 for bonding such polypeptides isfound, then polypeptides of increased size emulating the local region ofthe target macromolecule near the active bonding sites are generated andtested for bonding capability. Once acceptable bonding is attained, thenbonding of a known sample solution of the target macromolecule istested. Atomic force microscopy is then used to detect the bonding ofthe target macromolecule, and fluorescent labeling is discontinued. Thespecificity of the apparatus 1200 can then be assessed by testingadhesion of known false positive producing proteins. The structure ofthe apparatus 1200 is then adjusted to sterically hinder bonding of suchfalse positive producing proteins. In embodiments where conductors 1290and 1295 are provided, the bonding capabilities of the apparatus 1200can be adjusted by modulating voltage biases applied to such conductorsin order to improve the specific bonding affinity of the apparatus 1200for a target polypeptide macromolecule. Certain conductor compositionsenable charge carrier mobility predominantly in either p-doped orn-doped semiconductors. The specificity of this conductivity can befurther utilized to fabricate apparatus 1200 in which external voltagebiases applied to the conductors 1290 and 1295 can be used toselectively modify the bonding characteristics of the apparatus.

FIG. 23 shows an embodiment of a method 2300 for making amino aciddetection and identification apparatus 1600 as discussed above inconnection with FIG. 16. In a series of steps 2305, 2310, 2315 and 2320,the amino acid detection and identification apparatus 1600 isfabricated. In step 2305, a substrate 1634 formed from an electricallyinsulating composition is provided. In step 2310, a first comb 1602 isformed on the substrate 1634 comprising first tines 1604, 1606, 1608,1610, 1612, 1614, 1616 and 1618 formed from a first substantiallyinorganic composition selected from the group consisting of metals,semiconductors, insulators, and mixtures, said first tines havingselective affinity for bonding of a polypeptide. In step 2315, a secondcomb 1603 comprising second tines 1605, 1607, 1609, 1611, 1613, 1615,1617, 1619 and 1621 is formed from a second substantially inorganiccomposition selected from the group consisting of metals,semiconductors, insulators, and mixtures, said second tines havingselective affinity for bonding of a polypeptide. In step 2320, desirablycarried out simultaneously with steps 2310 and 2315, the first andsecond combs 1602 and 1603 are positioned on the substrate 1634 withtheir respective tines placed in mutually interwoven relationships at aspaced apart distance that is suitable for traversal by a polypeptidebonded to, or by multiple polypeptides located between and separatelybonded to, mutually adjacent first and second tines. The resulting aminoacid detection and identification apparatus 1600 can be used in a mannersimilar to that discussed in connection with the other apparatus above.

It will be recognized that the present teachings may be adapted to avariety of contexts consistent with this disclosure and the claims thatfollow. The apparatus disclosed herein may be designed for selectivebonding affinity with any amino acid—comprising molecules, ranging fromamino acids to macromolecules such as proteins. The substantiallyinorganic materials for fabrication of surfaces having bonding affinityfor such molecules broadly include metals, semiconductors and/orinsulators.

1. An apparatus, comprising: a first surface site layer having a firstdistal site end, wherein the first distal site end includes a firstsubstantially inorganic surface having a first chemical compositionselected from a group consisting of metals, semiconductors, insulators,and mixtures thereof, said first surface positioned within a polypeptidebonding region and having a selective bonding affinity for apolypeptide; a plurality of first interlayers between which said firstsurface site layer is interposed, wherein the first distal site end isdistanced from said first interlayers; first and second supports,wherein said first surface site layer and said first interlayers areinterposed between said first and second supports; and first and secondconductors provided on said first and second supports and havingrespective first and second distal conductor ends positioned within saidpolypeptide bonding region; wherein said conductors are capable ofapplying an external voltage potential across said polypeptide bondingregion.
 2. The apparatus of claim 1, further including: a second surfacesite layer having a second distal site end, wherein the second distalsite end includes a second substantially inorganic surface having asecond chemical composition selected from a group consisting of metals,semiconductors, insulators, and mixtures thereof, said second chemicalcomposition being different than said first chemical composition, saidsecond surface positioned within said polypeptide bonding region andhaving a selective bonding affinity for a polypeptide; and a pluralityof second interlayers between which said second surface site layer isinterposed; wherein the second distal site end is distanced from saidsecond interlayers, and said second surface site layer and said secondinterlayers are interposed between said first and second supports. 3.The apparatus of claim 1, in which said first chemical compositionincludes a metal selected from a group consisting of: palladium, gold,titanium, platinum, aluminum, magnesium, calcium, zirconium, vanadium,tantalum, chromium, molybdenum, tungsten, manganese, iron, cobalt,nickel, copper, silver, zinc, cadmium, gallium, indium, thalium, tin,lead, and mixtures thereof.
 4. The apparatus of claim 1, in which saidfirst chemical composition includes a semiconductor selected from agroup consisting of: gallium arsenide, aluminum gallium arsenide, indiumphosphide, indium gallium arsenide, indium gallium phosphide, indiumgallium arsenide phosphide, indium aluminum gallium arsenide, galliumnitride, indium nitride, aluminum nitride, aluminum gallium nitride,indium aluminum gallium nitride, gallium antimonide, indium antimonide,aluminum antimonide, aluminum gallium antimonide, indium aluminumgallium antimonide, indium arsenic antimonide, gallium aluminumantimonide, indium gallium antimonide, gallium arsenic antimonide, andmixtures thereof.
 5. The apparatus of claim 1, in which said firstchemical composition includes a metallic insulator composition selectedfrom a group consisting of: oxides, carbides, borides, nitrides,sulfides, and mixtures thereof.
 6. The apparatus of claim 5, in whichsaid metallic insulator composition includes a member selected from agroup consisting of: silicon nitride, silicon dioxide, aluminum oxide,zinc oxide, beryllium oxide, ferrite, zirconium oxide, boron carbide,silicon carbide, magnesium diboride, and mixtures.